Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite.
Downstream processing by biospecific chromatography of maltodextrin phosphorylase from Escherichia coli, overexpressed in E. coli, was substantially improved by a novel approach using ceramic hydroxyapatite. Wild-type and a less active mutant enzyme were purified from crude bacterial cell extracts in one efficient separation step that yielded phosphorylase in purity > 95% in at least 90% recoveries. At pH 6.9 and 25 degrees C, wild-type and mutant phosphorylases eluted from the hydroxyapatite column at a phosphate concentration of 0.4 M whereas calcium ions failed to displace the enzymes. The dynamic capacity for phosphorylase binding in the presence of bulk proteins was approximately 3 mg enzyme ml-1 matrix. The interaction of E. coli phosphorylase with hydroxyapatite seems to be mediated by surface amino groups, so that the bound enzyme retained almost full catalytic activity. Compared to the soluble enzyme, immobilization onto hydroxyapatite resulted in a more than 30-fold stabilization of wild-type phosphorylase against thermal and proteolytic inactivation and thus could improve the operational stability of phosphorylase during conversion of polysaccharide to glucose 1-phosphate.
Eis
C
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Institut für Lebensmitteltechnologie, Universität für Bodenkultur Wien (BOKU), Wein, Austria.
Griessler
R
R
Maier
M
M
Weinhäusel
A
A
Bock
B
B
Kulbe
K D
KD
Haltrich
D
D
Schinzel
R
R
Nidetzky
B
B
D002845:Chromatography / Q000379:methods; D017886:Durapatite / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005964:Glucosyltransferases / Q000235:genetics / Q000302:isolation & purification*; D008970:Molecular Weight; D016296:Mutagenesis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D017886:Durapatite; D005964:Glucosyltransferases; C033938:maltodextrin phosphorylase; D019286:Endopeptidase K